High-temperature-resistant and alkaline-resistant bacillus velezensis strain and application thereof in zearalenone degradation

Abstract

The invention relates to a bacillus velezensis strain resistant to high temperature and alkali, and belongs to the technical field of microbial detoxification and feed safety. The bacillus velezensis B.26 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.34475 is provided aiming at the problems that in the prior art, the thermal stability of zearalenone (ZEN) degrading enzyme is insufficient, the efficiency is low in an alkaline environment, and toxic metabolites are easily generated by traditional strains. CotA laccase and peroxide reductase Prx secreted by the strain can efficiently degrade ZEN and derivatives alpha-zearalenol and beta-zearalenol, the products are low-toxicity C17H24O4 and C12H16O4, and harmful by-products such as alpha-ZEL / beta-ZEL and the like do not exist. The strain and recombinase thereof are suitable for a high-temperature alkaline environment for feed processing, and can be applied to biological detoxification of ZEN pollution in food and feed on a large scale through an immobilized carrier.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detoxification and feed safety, and more particularly to a thermophilic and alkaline-resistant Bacillus velezensis strain and its application in the degradation of zearalenone. Background Art

[0002] Zearalenone (ZEN) is a class of estrogenic mycotoxins produced by Fusarium spp. that widely contaminates grains such as corn and wheat, as well as their processed products. Its chemical structure is stable and difficult to decompose at high temperatures (e.g., 120°C) and under conventional food processing conditions, resulting in long-term residues in the food and feed chain. ZEN binds to estrogen receptors by mimicking 17β-estradiol, interfering with endocrine function and causing reproductive damage, immunosuppression, and oxidative stress, posing a serious threat to animal (especially pigs) and human health. Epidemiological surveys show that the global ZEN contamination rate in pig feed exceeds 40%, and ZEN concentrations in processed grain products in high-humidity areas are significantly increased, further exacerbating food safety risks.

[0003] At present, the detoxification methods for ZEN mainly include physical adsorption, chemical treatment and biodegradation. Although physical methods (such as montmorillonite adsorption) are simple to operate, they have problems such as low adsorption specificity and easy loss of nutrients. Chemical methods (such as ozone oxidation and photocatalytic degradation) can effectively destroy the ZEN structure, but may generate byproducts of unknown toxicity, and have high equipment requirements, making it difficult to apply on a large scale. Biological detoxification has become a research hotspot due to its high efficiency and environmental friendliness. Among them, enzymatic degradation has attracted much attention due to its high specificity and mild reaction conditions. However, the existing technology still faces the following bottlenecks:

[0004] 1. Insufficient enzyme thermal stability: Most reported ZEN-degrading enzymes (e.g., lactone hydrolase ZHD101 and fungal laccases) are easily inactivated at high temperatures (>60°C), making them difficult to adapt to the requirements of feed processing (e.g., pelleting temperatures of 55-80°C) or food sterilization processes. For example, the fungal laccase PpLac1 exhibits optimal activity at pH 3.0-5.0, but its efficiency decreases significantly in neutral or alkaline environments, limiting its application in complex matrices.

[0005] 2. Conflict between degradation efficiency and safety: While some microorganisms (such as certain Bacillus species) can degrade ZEN, their metabolic pathways may produce more toxic derivatives (such as α-ZEL and β-ZEL), increasing the risk of secondary contamination. Furthermore, the degradation efficiency of traditional strains is limited by environmental tolerance (such as pH and metal ion sensitivity), making it difficult to maintain stable performance in industrial scenarios.

[0006] 3. High cost of enzyme expression and preparation: The expression level of wild-type enzymes is low, and heterologous expression systems (such as Escherichia coli) often face problems such as inclusion body formation and loss of enzyme activity, resulting in high costs for industrial production.

[0007] In recent years, researchers have attempted to address these issues by improving the thermal stability of enzymes or optimizing expression systems through protein engineering. However, these modified enzymes often compromise catalytic efficiency or environmental adaptability. For example, after improving the thermal stability of ZHD101 through site-directed mutagenesis, its degradation activity decreased by approximately 30% compared to the wild type. Furthermore, while existing immobilized carriers (such as calcium alginate microspheres) can improve the operational stability of enzymes, the binding efficiency of the carrier and the enzyme and their reusability still need to be improved.

[0008] In this context, the development of new ZEN-degrading enzymes and their application technologies that are highly efficient, thermally stable and safe have become a key breakthrough direction in solving the problem of mycotoxin contamination in food and feed.

[0009] In addition, in the alkaline processing environment (pH>10) of the feed industry, existing ZEN biodegradation technology faces multiple challenges:

[0010] Poor alkaline resistance of strains: The activity of degrading bacteria such as Bacillus Velezii drops sharply when pH>9 (e.g., degradation rate 70% at pH 7.0 → <30% at pH>9) because high pH destroys cell membrane structure, inhibits enzyme activity (key enzymes have an optimal pH of neutral), and interferes with metabolic pathways.

[0011] Low system stability: High pH leads to raw material denaturation, viscosity fluctuation, obstructed dissolved oxygen transfer, accumulation of organic acid metabolic byproducts, and browning of a single carbon source (such as glucose) exacerbating carbon starvation;

[0012] Difficulty in domestication and regulation: Traditional pH gradient domestication requires 4-6 weeks and the strain is prone to degeneration. Preservatives (glycerol, etc.) and metal ion precipitation aggravate damage. Insufficient coordinated control of pH / dissolved oxygen / carbon source causes local pH mutations (±0.5), shear damage, and carbon supply imbalance.

[0013] The existing methods fail to integrate the development of alkali-resistant strains, metabolic regulation and process optimization, resulting in degradation rates <50%, cycles >48 hours, and bacterial survival rates (OD600 decrease >60%), which seriously restricts industrial applications. Summary of the Invention

[0014] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0015] In order to achieve these purposes and other advantages according to the present invention, a high-temperature and alkaline-resistant Bacillus velezensis strain is provided, which is Bacillus velezensis B.26 strain, classified and named Bacillus velezensis. It is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms (CGMCC) with a deposit number of CGMCC No. 34475. The deposit date is May 9, 2025, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain can degrade zearalenone and its derivatives α-zearalenol and β-zearalenol.

[0016] Provided is an application of the Bacillus velez in degrading zearalenone, wherein the application is carried out at a temperature of 50-80°C and a pH of 7.0-12.0, and the reaction time is 1-24 hours, so that zearalenone is degraded into a low-toxic metabolite C 17 H 24 O4 and C 12 H 16 O4, and no α-zearalenol or β-zearalenol was produced.

[0017] Provided is an application of the Bacillus Velezii in preparing an enzyme for degrading zearalenone.

[0018] Preferably, the enzyme that degrades zearalenone is a recombinant CotA laccase, the amino acid sequence of which is shown in SEQ ID NO: 3, and the nucleotide sequence of the recombinant CotA laccase is shown in SEQ ID NO: 1.

[0019] Preferably, the enzyme that degrades zearalenone is a recombinant peroxide reductase Prx, the amino acid sequence of which is shown in SEQ ID NO: 4, and the nucleotide sequence of the recombinant peroxide reductase Prx is shown in SEQ ID NO: 2.

[0020] Provided is a method for degrading zearalenone, comprising the following steps:

[0021] contacting the recombinant CotA laccase or the recombinant peroxide reductase Prx with a substrate containing zearalenone;

[0022] The reaction is carried out at a temperature of 50-80° C. and a pH of 7.0-12.0 for 1-24 hours to degrade ZEN into low-toxic metabolites.

[0023] Provided is a detoxification agent composition, comprising the recombinant CotA laccase and / or the recombinant Prx, and a pharmaceutically or feed-acceptable carrier; the detoxification agent composition is used for the degradation of zearalenone in food or feed, wherein the carrier is calcium alginate microspheres, montmorillonite-based materials or nanoparticles, which are used for enzyme immobilization or sustained release.

[0024] A method for fermenting and degrading zearalenone by Bacillus velezensis, which is applied to alkaline feed processing, is provided, comprising the following steps:

[0025] Step 1: crushing the zearalenone-contaminated feed raw material to a particle size of 0.5-1.0 mm, and mixing it with sterile water at a mass ratio of 1:5 to obtain a pretreated feed suspension;

[0026] Step 2: Inoculate Bacillus velezensis CGMCC No. 34475 into an activation medium containing 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride, adjust the initial pH to 8.5 with sodium bicarbonate buffer, and incubate the culture in a 37°C constant temperature shaker at 180 rpm for 12 hours;

[0027] Step 3: Inoculate the activated bacterial solution into the pretreated feed suspension at a volume ratio of 10%, add sucrose with a mass concentration of 0.3%-0.5% as an induced carbon source, and use a sodium carbonate solution with a mass concentration of 5% to adjust the initial pH of the fermentation system to 10.5;

[0028] Step 4: The fermentation system was placed in a sealed fermentation tank, the temperature in the fermentation tank was controlled at 37°C, sterile air was introduced to maintain the dissolved oxygen concentration at 30%-40%, and the pH of the fermentation liquid was monitored in real time by an online pH probe and automatically supplemented with a 5% mass concentration of sodium carbonate solution to maintain the pH of the fermentation liquid in the range of 10.5-11;

[0029] After 24 hours of fermentation under continuous stirring at 180 rpm, the fermentation was terminated;

[0030] Step 5: After solid-liquid separation of the fermented material, the solid material is dried at 60° C. with hot air to a moisture content of less than 12%, thereby obtaining a finished feed product after degradation treatment.

[0031] Preferably, the activation culture of Bacillus velezinis adopts a staged pH adaptive acclimation operation, the specific steps of which include:

[0032] Under the condition that the initial pH of the activation medium was adjusted to 8.5, the culture was carried out in a constant temperature shaker at 37°C and 180 rpm for 8 hours;

[0033] The pH of the culture system was gradually raised to 10.0 by aseptically adding 5% sodium carbonate solution, and the culture temperature was maintained at 37°C for another 4 hours.

[0034] During the pH increase process, when the OD600 value of the culture solution dropped by more than 15%, 0.1% trehalose was immediately added as a cell membrane protectant.

[0035] After completing the two-stage culture, the bacterial solution was inoculated back into fresh activated culture medium at a volume ratio of 15%, and cultured for another 3 hours at pH 10.0 to obtain an alkali-resistant acclimated strain.

[0036] Preferably, in step 3, the fermentation induction operation adopts a phased supply strategy of a composite carbon source, and the specific steps include:

[0037] During the fermentation period of 0-12 hours, the stirring speed was maintained at 180 rpm, and sucrose with a mass concentration of 0.3% was continuously added as the basic carbon source;

[0038] During the fermentation period of 12-24 hours, a complex of 0.1% inulin and 0.05% sodium citrate was added simultaneously, wherein the mass ratio of inulin to sodium citrate was 2:1;

[0039] When the viscosity of the fermentation broth rises to 2000 cP, a 0.2% mass concentration of β-cyclodextrin solution is pulse-injected into the fermentation broth, and the injection volume each time is 1% of the total volume of the fermentation broth;

[0040] The cumulative amount of β-cyclodextrin added does not exceed 0.1% of the dry matter mass of the fermentation liquid;

[0041] During the fermentation process, the ATP content of the bacteria was detected every 6 hours. When the ATP content was lower than 5 μmol / gDCW, the carbon source supply was immediately terminated and the cooling program was started.

[0042] The present invention has at least the following beneficial effects:

[0043] First, Bacillus velez strain B.26 (CGMCC No. 34475) can efficiently degrade zearalenone (ZEN) and its derivatives α-zearalenol and β-zearalenol. The enzymes it secretes can convert ZEN into low-toxic metabolites C 17 H 24 O4 and C 12 H 16 O4, and no harmful byproducts such as α-ZEL / β-ZEL. This strain is suitable for the high temperature (50-80°C) and alkaline (pH 7.0-12.0) environments of feed processing. It solves the problems of insufficient thermal stability of degradation enzymes in existing technologies, low efficiency in alkaline environments, and the easy generation of toxic metabolites by traditional strains, providing a safe and efficient microbial resource for food and feed detoxification.

[0044] Second, Bacillus velezensis B.26 or its functionally equivalent mutants have broad applications in ZEN degradation. By secreting CotA laccase and peroxide reductase Prx, this strain maintains high degradation activity under high-temperature alkaline conditions, achieving a degradation rate exceeding 89%. Compared to physical adsorption and chemical treatment methods, it offers strong specificity, no secondary contamination, and can be applied on an immobilized carrier for large-scale application, significantly improving the efficiency of bioremediation of ZEN contamination in feed and food, safeguarding food safety and animal health.

[0045] Third, the genome of Bacillus velezensis strain B.26 contains genes encoding CotA laccase and peroxide reductase Prx. CotA laccase has a sequence identity of ≥66.12% with CotA from Bacillus licheniformis, while Prx has a sequence identity of ≥42.65% with the peroxide reductase from Acinetobacter hospitalis Y1. These two enzymes work synergistically to efficiently degrade ZEN. CotA laccase achieves a degradation rate exceeding 90% in 6 hours at 70°C and pH 8.0, while Prx remains active at pH 11.0. This addresses the challenges of existing enzymes, such as poor thermal stability and low efficiency in alkaline environments, and broadens their application scenarios.

[0046] Fourth, the amino acid sequence of the recombinant CotA laccase is shown in SEQ ID NO: 3 or its variant (≥90% sequence identity and retained activity), which has the ability to efficiently degrade ZEN. The enzyme is stable at 70°C, with 80% activity remaining after 1 hour, and is affected by Cu 2+ The degradation rate can reach 96% when promoted by metal ions such as β-lactamase. Its heterologous expression system (such as Escherichia coli) can achieve high yields (710 mg / L), and the variant design increases the environmental adaptability of the enzyme, providing a low-cost, highly active biocatalyst for industrial production.

[0047] Fifth, the recombinant peroxide reductase Prx, whose amino acid sequence is shown in SEQ ID NO: 4 or a variant thereof (≥90% sequence identity and retained activity), can effectively degrade ZEN. The enzyme retains >80% activity in the pH range of 9.0-12.0, and 75% activity remains after treatment at 70°C for 1 hour, making it suitable for alkaline industrial environments. When it works synergistically with CotA laccase, the ZEN degradation rate can reach 98.2%, and the product has no estrogenic activity, which compensates for the insufficient catalytic efficiency of a single enzyme and improves the stability and safety of the detoxification system.

[0048] Sixth, a fermentation method for alkaline feed processing involves pretreatment with pulverization, staged pH control, and the supply of a complex carbon source. Under conditions of pH 10.5-11 and 37°C for 24 hours, the method achieves a ZEN degradation rate of 92%. By optimizing strain activation conditions and fermentation parameters, this method overcomes the challenges of traditional alkaline fermentation, such as insufficient alkaline tolerance, dissolved oxygen limitations, and low carbon source utilization efficiency. This method ensures bacterial activity and metabolic stability, making it suitable for high-humidity, high-alkaline feed processing environments and achieving efficient biodetoxification of ZEN-contaminated feed.

[0049] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Figures 1(A) are liquid chromatography analysis and degradation effect diagrams of cell-free supernatant, wherein 1(A) is liquid chromatography analysis of degradation products of zearalenone (ZEN) by Bacillus velezinoffii B.26; Figure 1 (B) Degradation effect of ZEN by cell-free supernatant and bacterial cells over 24 hours;

[0051] Figure 2 This is an analysis of the degradation characteristics of zearalenone (ZEN) by Bacillus velez B.26, Figure 2 (A) Effect of pH on ZEN degradation (37°C, 24 h, ZEN concentration 2 μg / mL); Figure 2 (B) Effect of degradation temperature on ZEN degradation (pH 10.0, 24 h, ZEN concentration 2 μg / mL); Figure 2 (C) Effects of metal ions on ZEN degradation by strain B.26 (70°C, pH 10.0, 24 h, ZEN concentration 2 μg / mL, metal ion concentration 10 mM); Figure 2 (D) Effect of ZEN concentration on degradation (70°C, pH 10.0, 24h); Figure 2 (E) Effects of different treatments on ZEN degradation (70°C, pH 10.0, 24 h); Figure 2 (F) Effect of degradation time on ZEN degradation (70°C, pH 10.0, ZEN concentration 2 μg / mL) (p < 0.05);

[0052] Figure 3 This is the whole genome identification analysis of strain B.26; Figure 3 (A) is the whole genome identification result of strain B.26; Figure 3 (B) is the species annotation statistics based on the NR database;

[0053] Figure 4 BlastP alignment analysis, where Figure 4 (A) BlastP alignment analysis based on annotated protein coding databases shows that the amino acid sequence identity (Identities%) between GM03635 protein and Bacillus licheniformis CotA laccase is 66.12%; Figure 4 (B) BlastP alignment analysis based on annotated protein coding databases showed that the amino acid sequence identity between GM03860 protein and the peroxide reductase (Prx) of Acinetobacter nosocomialis Y1 strain was 42.65%; Figure 4 (C) Annotation of the conserved domains of CotA protein; Figure 4 (D) Annotation of the conserved domains of Prx proteins;

[0054] Figure 5 The SDS-PAGE analysis of CotA and Prx and their molecular docking simulation with ZEN; Figure 5 (A) SDS-PAGE analysis of CotA, lane M: protein marker (Thermo Fisher Scientific, USA); lane 1: BL21 (pET28a-CotA) bacterial pellet; lane 2: BL21 (pET28a-CotA) bacterial lysate supernatant; lanes 3-4: Ni-NTA-purified CotA protein; Figure 5 (B) SDS-PAGE analysis of Prx, lane M: protein marker (Beijing Solebao Company); lane 1: BL21 (pET28a-Prx) bacterial pellet; lane 2: BL21 (pET28a-Prx) bacterial lysate supernatant; lanes 3-4: Ni-NTA-purified Prx protein; Figure 5 (C) Molecular docking simulation of CotA and ZEN shows that ZEN binds to the side chains of serine (Ser186), lysine (Lys180), and arginine (Arg248) of CotA through six hydrogen bonds; Figure 5 (D) Molecular docking simulation of Prx and ZEN shows that ZEN binds to the aspartic acid (Asp93, Asp94) and lysine (Lys97) side chains of Prx through three hydrogen bonds;

[0055] Figure 6 The enzymatic characteristic parameters analysis of CotA and Prx in degrading zearalenone (ZEN) were analyzed. Figure 6 (A) Detoxification effect of CotA on ZEN at different incubation times; Figure 6(B) Effect of ZEN concentration on CotA degradation (70°C, pH 8.0, 6h); Figure 6 (C) Effect of temperature on CotA degradation (pH 8.0, 6 h, ZEN concentration 5 μg / mL); Figure 6 (D) Effect of pH on CotA degradation (70°C, 6 h, ZEN concentration 5 μg / mL); Figure 6 (E) Effects of metal ions on CotA degradation (70°C, pH 8.0, 6 h, ZEN concentration 5 μg / mL); Figure 6 (F) The detoxification effect of Prx on ZEN at different incubation times; Figure 6 (G) Effect of ZEN concentration on Prx degradation (70°C, pH 11.0, 6h); Figure 6 (H) Effect of temperature on Prx degradation (pH 9.0, 6 h, ZEN concentration 5 μg / mL); Figure 6 (I) Effect of pH on Prx degradation (70°C, 6 h, ZEN concentration 5 μg / mL); Figure 6 (J) Effects of metal ions on Prx degradation (70°C, pH 11.0, 6 h, ZEN concentration 5 μg / mL);

[0056] Figure 7 Analysis of degradation products of zearalenone (ZEN) by CotA; Figure 7 (A) is the mass spectrum of ZEN blank control; Figure 7 (B) is the mass spectrum of the system containing only CotA degradation enzyme;

[0057] Figure 8 This is the analysis of the degradation products of zearalenone (ZEN) in the CotA treatment group and Prx, among which, Figure 8 (A) is the mass spectrum of the ZEN positive control in the CotA-treated group; 8(B) is the mass spectrum of the ZEN blank control;

[0058] Figure 9 Analysis of degradation products of zearalenone (ZEN) by Prx, among which, Figure 9 (A) is the mass spectrum of the system containing only Prx degrading enzyme; Figure 9 (B) is the mass spectrum of the ZEN positive control in the Prx-treated group;

[0059] Figure 10 is the secondary mass spectrum, where Figure 10 (A) is the MS / MS spectrum of ZEN; Figure 10 (B) is the MS / MS spectrum of the main degradation products under CotA treatment;

[0060] Figure 11is the secondary mass spectrum of the main degradation products under Prx treatment;

[0061] Figure 12 Chemical structures of the main degradation products: CotA-treated products (C 17 H 24 O4) and Prx treatment products (C 12 H 16 O4). DETAILED DESCRIPTION

[0062] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0063] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0064] 1. Laboratory test

[0065] 1. Materials and Methods

[0066] 1.1 Materials, strains, and plasmids

[0067] HPLC-grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (Beijing). Analytical-grade zearalenone (ZEN) standard was purchased from Prebond (Shandong). Key molecular biology reagents, including isopropyl-β-D-thiogalactopyranoside (IPTG), SDS-PAGE gel kit, and custom DNA oligonucleotides for PCR amplification, were provided by Beyotime Biotechnology (Shanghai). Bacillus velezensis B.26 was stored frozen at −80°C. For genetic manipulation, Escherichia coli DH5α and BL21 (DE3) competent cells (Sangon Biotechnology, Shanghai) were used as cloning and expression hosts, respectively, and the pET28a expression vector was previously stored in the laboratory resource library.

[0068] 1.1.1 Screening of ZEN-degrading bacteria

[0069] A basic mineral salt medium (MSM) was designed with ZEN as the sole carbon source for strain screening and isolation.

[0070] Initial screening: Weigh 5 g of the collected sample, add 25 mL of sterile water, and shake at 37°C for 3 hours. Let it stand at room temperature for 1 hour, then remove the supernatant for later use. Add the supernatant to MSM (containing 2 μg / mL ZEN) at a 5% (v / v) inoculum and incubate at 37°C, 180 rpm, and shake for 2 days for enrichment. Add an equal amount of chromatography-grade methanol to terminate the reaction, vortex thoroughly, and centrifuge at 4°C, 12,000 rpm, for 10 minutes. The supernatant is filtered through a 0.22 μm organic phase filter into a liquid phase vial. High-performance liquid chromatography (HPLC) is used to determine the residual ZEN content. A fresh sterile culture medium containing the same ZEN concentration is used as a control for calculating the degradation rate.

[0071] Isolation and rescreening: Fermentation broth with significant degradation effects was diluted with sterile water in a gradient manner, and 100 μL was aspirated and spread onto LB solid medium. The culture was then incubated at 37°C for 2 days. Single colonies with different morphologies and good growth were picked and added to LB liquid medium containing a final concentration of 2 μg / mL ZEN. The culture was shaken at 37°C and 180 rpm for 24 hours. An equal amount of chromatography-grade methanol was added to terminate the reaction. Subsequent steps were the same as the initial screening steps. The remaining ZEN content was detected by high-performance liquid chromatography (HPLC). The effective bacterial liquid was co-cultured with ZEN and passaged three times. Three replicates were set up for each experimental group. Strains with good degradation ability on all three occasions were streaked onto LB solid medium and incubated at 37°C for 1 day to obtain a single strain and preserve it.

[0072] MSM medium (1 L): Na2HPO4 2800 mg, (NH4)2SO4 500 mg, CuCl2·2H2O 0.001 mg, H3BO3 0.03 mg, FeSO4·7H2O 0.2 mg, MnCl2·4H2O 0.003 mg, NiCl2·6H2O 0.002 mg, KH2PO4 1000 mg, Na2EDTA 0.5 mg, CoCl2·6H2O 0.02 mg, ZnSO4·7H2O 0.01 mg, Na2MoO4·2H2O 0.003 mg were added to 1 L of deionized water and the pH was adjusted to 7.2. Autoclave at 121°C for 15 min. When cooled to 50°C, 0.5 mL of 2000×CaCl2 was added. 2+ &Mg 2 + Solution, mix well and set aside.

[0073] 1.2 Detection of ZEN by HPLC

[0074] ZEN concentration was analyzed using an Agilent 1260 Infinity HPLC (USA) using a reversed-phase C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase for ZEN was 70% acetonitrile in water. The excitation and emission wavelengths of the fluorescence detector (FLD) were 274 nm and 440 nm, respectively. The mobile phase flow rate was 1 mL / min, and the column temperature was set at 28°C.

[0075] 1.3 Degradation characteristics of ZEN by Bacillus velezensis B.26

[0076] Strain B.26 stored at -80°C was activated in 3 mL of LB broth and incubated at 37°C in a shaker at 180 rpm for 16 hours. The culture was inoculated at a 1% (v / v) inoculum into 2 mL of LB broth containing 2 μg / mL ZEN and incubated for 24 hours. Mycotoxin concentrations were then quantified by HPLC as described in Section 1.2.

[0077] 1.3.1 Effects of Bacillus velezensis B.26 Cell Components on ZEN Degradation

[0078] Bacillus velezensis B.26 was cultured in LB broth at 37°C for 24 hours. After incubation, the samples were centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was filtered through a 0.22 μm filter and transferred to a sterile tube. The pellet was washed twice with 1×PBS (pH 7.4) and then resuspended with an equal volume of 1×PBS. To prepare cell lysate, the washed pellet was lysed using an ultrasonic cell disruptor at 4°C for 10 minutes, with the ultrasonic power set to 325 W and a pulse on for 2 seconds and off for 3 seconds as one cycle. Subsequently, the cell lysate was collected by centrifugation at 8,000 rpm for 15 minutes at 4°C. ZEN was added to the supernatant, cell suspension, and cell lysate to a final concentration of 2 μg / mL and incubated at 70°C for 24 hours. After incubation, the residual amount of ZEN was determined using HPLC.

[0079] 1.3.2 Effects of SDS, Proteinase K, and Supernatant Heat Treatment on ZEN Degradation

[0080] Three different biochemical treatments were introduced into the supernatant of strain B.26, including 1% sodium dodecyl sulfate (SDS) and 1 mg / mL proteinase K. Another portion of the filtered supernatant was heat denatured at 100°C for 15 minutes. Following these treatments, all samples were supplemented with 2 μg / mL ZEN and incubated at 70°C and 180 rpm for 24 hours. Following incubation, ZEN levels were quantified using HPLC.

[0081] 1.3.3 Effect of metal ions on supernatant ZEN degradation

[0082] The cell-free supernatant of Bacillus velezensis B.26 was aseptically aliquoted into sterile tubes, and each sample was spiked with 10 mM solutions of NaCl, KCl, LiCl, CaCl2, MnCl2, MgSO4, CuSO4, ZnSO4, FeSO4, and Fe2(SO4)3. After the addition of the metal cofactors, all samples were spiked with ZEN to a final concentration of 2 μg / mL. The samples were then incubated at 70°C and 180 rpm for 24 hours, and the ZEN degradation efficiency was quantified by HPLC.

[0083] 1.3.4 Effects of mycotoxin concentration, pH, temperature, and incubation time on the degradation rate of ZEN in supernatant

[0084] To systematically investigate mycotoxin degradation parameters, ZEN was added to the supernatant of strain B.26 at final concentrations of 1, 2, 5, 10, 20, 30, and 40 μg / mL and then degraded at 70°C for 24 hours. To examine the effect of pH, the supernatant was adjusted to a pH of 5-12, and ZEN was added to the samples at a final concentration of 2 μg / mL. The samples were then incubated at 70°C for 24 hours. To assess thermal stability parameters, ZEN was added to the supernatant of strain B.26 at a final concentration of 2 μg / mL and incubated at different temperatures (28-75°C) for 24 hours. To investigate the effect of incubation time, ZEN was added to the supernatant of strain B.26 at a final concentration of 2 μg / mL and incubated at 70°C for different times (4-60 hours). Finally, ZEN degradation was analyzed by HPLC.

[0085] 1.4 Sequencing, assembly, annotation, genome comparison, and identification of enzymes involved in ZEN degradation

[0086] The genome of strain B.26 was sequenced by Novogene (Beijing) using the PacBio RS II platform in HiFi (high-fidelity) circular consensus sequencing (CCS) mode, and long reads (≥10kb) with an accuracy of >99.9% were generated through sub-read self-correction. Based on the above sequencing data, Canu (https: / / github.com / marbl / canu / , v2.0) was used for de novo genome assembly to generate preliminary contigs representing the genome structure. Subsequently, Racon (v1.4.13) was used to perform three rounds of iterative error correction on the assembly draft using third-generation sequencing reads, and then Pilon (v1.22) was used in combination with second-generation short reads for three additional polishing cycles to finally obtain an optimized genome assembly.

[0087] Short reads generated by the HiSeq platform were assembled into contigs using SOAPdenovo v.2.04, and PacBio reads were assembled using the RS-HGAP-Assembly3 protocol in the SMRT Analysis v2.2.0 pipeline. Functional annotation was performed by aligning all protein sequences with public databases including COG (Orthologous Groups), GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), NR (Non-Redundant Protein Database), and SwissProt using BLASTp with an E value of 10 -5 Comparative genomic analysis was performed using the BLASTP algorithm via the NCBI web interface with default parameters (E-value threshold 10, BLOSUM62 matrix). The NCBI non-redundant laccase database was screened in parallel to identify conserved catalytic domains. Sequence similarity thresholds for functional annotation validation were set at ≥30% identity and ≥80% coverage.

[0088] The identification of candidate enzymes was performed by a systematic bioinformatics workflow utilizing the laboratory-curated Mycotoxin Degrading Enzyme Database (MDED v2.1). The whole genome sequence of B.26 was aligned to the MDED reference sequence by local BLASTp analysis (E-value cutoff: 1e-50). The enzymes were analyzed using InterProScan v5.62-94.0.

[0089] (https: / / www.ebi.ac.uk / interpro / ) for primary sequence analysis, and the Pfam and SMART protein signature databases were queried.

[0090] 1.5 Molecular docking analysis

[0091] ZEN was selected as a ligand for molecular docking, and its three-dimensional structure was retrieved from the PubChem database. The tertiary structure of the target enzyme was constructed using homology modeling using the SWISS-MODEL platform (CotA PDB: 2WSD; Prx PDB: 7KQ6). Template screening prioritized sequences with >20% sequence identity and >85% structural coverage to ensure model fidelity. Structural verification was performed using topological analysis in PyMOL 2.5 to confirm correct folding and active site geometry.

[0092] Molecular docking simulations were performed in AutoDock Vina 1.2.0 using semiflexible ligand parameters. Receptor preparation included removal of crystallographic waters, addition of polar hydrogens, assignment of Gasteiger charges, and optimization of torsion parameters, generating a protein file in PDBQT format. Ligand preprocessing included conformational energy minimization, partial charge calculation, and definition of rotatable bonds. A grid box was defined containing the catalytic pocket. The grid spacing and number of points were kept at the default settings. Electrostatic and desolvation potential maps were generated using AutoGrid4.

[0093] A Lamarckian genetic algorithm generated 10 binding conformations through 2.5 million energy evaluations. After processing the docking parameter profile in AutoDock4, the conformations were ranked by calculated binding free energy. The conformation with the lowest binding energy was selected as the primary binding mode. Finally, the docking interface was visualized and optimized for aesthetics using molecular graphics software.

[0094] 1.6 Cloning of the degradation enzyme and its heterologous expression in Escherichia coli (BL21)

[0095] Based on the comparison results, the next step of cloning and heterologous expression was carried out. Genomic DNA was extracted from strain B.26 using the Tiangen Bacterial DNA Kit (Tiangen Biochemical, Beijing), and the target gene was amplified by PCR using the primers listed in Table 1. The thermal cycling parameters were set according to the instructions. The amplified products were electrophoresed on a 1% agarose gel and purified using the Meiji HiPure Gel Purification DNA Mini Kit (Meiji Biotechnology, Guangzhou).

[0096] Table 1 Primer sequence list

[0097]

[0098] Prx was restricted using BamHI / HindIII, CotA was restricted using BamHI / EcoRI, and the pET28a(+) vector was digested with the corresponding enzymes. The digested fragments were gel-purified and ligated into the linearized vector using the MutExpress II Rapid Mutagenesis Kit V2 (Weizan Bio). The recombinant plasmids were transformed into Escherichia coli DH5α competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. Positive clones were verified by colony PCR and Sanger sequencing (Shanghai Biotechnology Co., Ltd.), and constructs with a sequence identity of 100% were selected for subsequent transformation into BL21(DE3).

[0099] For protein expression, the recombinant BL21(DE3) strain was cultured in LB medium containing 50 μg / mL kanamycin at 37°C and 180 rpm. When the OD600 reached 0.6, protein production was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), followed by overnight incubation at 10°C and 120 rpm to promote soluble expression. The cell pellet was collected by centrifugation (4000 × g, 10 minutes) for subsequent inclusion body isolation and enzymatic characterization.

[0100] The structures of CotA and Prx were predicted using the SWISS-Model server (https: / / swissmodel.expasy.org / ), and molecular docking simulations were performed using Autodock. Comparative sequence alignment in DNAMAN further elucidated the evolutionary conservation of homologous proteins.

[0101] 1.7 Purification of recombinant CotA and recombinant Prx

[0102] Cell pellets containing recombinant Prx and CotA proteins were collected from 50 mL of culture by centrifugation (12,000 × g, 20 minutes, 4°C) and washed three times with ice-cold phosphate-buffered saline (PBS, pH 7.4). Protein solubilization was achieved by incubation with lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM pH 8.0 Tris-HCl, 1 mM protease inhibitor cocktail) under continuous rotation, followed by lysis using a Scientz ultrasonic cell disruptor for 10 minutes, with the ultrasonic power set to 600 W and a pulse on cycle of 2 seconds and off cycle of 3 seconds.

[0103] The lysate was clarified by ultracentrifugation (12,000 × g, 30 minutes, 4°C), and the supernatant fraction was loaded onto a Ni-NTA column (5 mL bed volume). After binding, the column was washed for 10 column volumes with wash buffer containing 20 mM imidazole. The target protein was eluted using a step gradient of elution buffer containing 250 mM imidazole, and 2 mL fractions were collected.

[0104] Protein purity and molecular weight were verified by SDS-PAGE analysis using a discontinuous buffer system (10% separating gel, 5% stacking gel) under reducing conditions. The gel was stained with Coomassie Brilliant Blue R-250 and destained with ethanol:acetic acid:water (4:1:5 v / v) to visually confirm that the recombinant protein band was at the predicted molecular weight.

[0105] 1.8 Analysis of CotA enzymatic properties

[0106] This study investigated the enzymatic properties, including optimal incubation time, optimal reaction temperature, thermal stability, optimal reaction pH, pH stability, the effect of metal ions, and enzyme kinetics. + , K + 、Li + , Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ 、Mn 2+ 、Fe 3+The enzymatic properties of CotA and Prx were determined using 500 μL Eppendorf tubes containing 20 μg / mL of recombinant enzyme and 5 μg / mL of ZEN.

[0107] 1.9 Identification of ZEN degradation products

[0108] Samples containing 2 μg / mL ZEN were incubated with CotA and Prx for 6 hours under their respective optimal temperature and pH conditions (as described in Section 1.8). The reaction was terminated by adding an equal volume of methanol. The experimental setup included three replicates of a blank control (no enzyme system), a negative control (no ZEN system), and a ZEN standard solution. ZEN products from the reaction with CotA and Prx were analyzed using a Dionex Ultimate 3000 UHPLC system coupled with a Q Exactive mass spectrometer equipped with a heated electrospray ionization (HESI) source. Chromatographic separation was performed on a ZORBAX Eclipse Plus C18 column (3.0 mm × 150 mm, 1.8 μm) at 40°C with an injection volume of 2 μL and a flow rate of 0.3 mL / min. The mobile phase consisted of 0.1% formic acid in water and acetonitrile. Mass spectrometric detection was performed in negative ionization mode (ESI-), and data acquisition and processing were performed using Xcalibur software (Thermo Fisher Scientific).

[0109] 1.10 Statistical Analysis

[0110] All experiments were performed in triplicate using three independent experimental groups. Statistical analysis was performed using one-way ANOVA. Statistically significant differences are indicated by lowercase letters; the same letters indicate no significant difference between groups (p>0.05); different letters indicate a significant difference between groups (p<0.05).

[0111] 2. Test results

[0112] 2.1 ZEN degradation characteristics of strain B.26

[0113] The degradation ability of strain B.26 on ZEN is as follows Figure 1 As shown in A, strain B.26 had a high degradation ability of 2 μg / mL ZEN within 24 hours. Figure 1 B shows the active ingredients involved in the degradation of ZEN in strain B.26. Compared with the degradation effect of 91.64%±0.24% of the bacterial solution, the degradation ability of the resuspended bacteria was significantly reduced to 36.35%±0.42%. The degradation of ZEN by the inactivated bacteria group and the cell content group was reduced by about 45%, while the degradation efficiency of ZEN by the supernatant was 89.82%±1.24%, indicating that the active ingredients were mainly present in the supernatant.

[0114] The supernatant was then analyzed for degradation properties, e.g. Figure 2 As shown in Figure A, 36.87% ± 0.57% of ZEN could be degraded at pH 10. Compared with the acidic environment, strain B.26 showed higher degradation activity under alkaline conditions. Figure 2 As shown in Figure B, with the increase of temperature, the degradation rate of ZEN showed a trend of first increasing and then decreasing. Under the condition of pH 10.0, 70℃ was the optimal degradation temperature, and the degradation rate was 83.40%±1.13%.

[0115] Figure 2 C shows the effect of metal ions on ZEN degradation. For ZEN degradation, Cu 2+ The degradation activity was significantly enhanced, with a degradation rate of 93.13% ± 0.62%, while Fe 3+ The degradation capacity of the supernatant was inhibited to the greatest extent, resulting in a decrease of 43.21% ± 0.75% in the degradation rate. 2+ Mg 2+ In the presence of Cu ions, the reaction was inhibited to less than 60%. These results indicate that the thermostable enzyme in the supernatant of strain B.26 may be involved in the degradation of ZEN, and that 2+ It can enhance the degradation activity of these enzymes.

[0116] Figure 2 Figure D shows the effect of mycotoxin concentration. At a concentration of 1 μg / mL, the supernatant of strain B.26 was able to degrade 86.58% ± 1.67% of ZEN within 24 hours. At a concentration of 40 μg / mL, the supernatant still retained 77.69% ± 0.96% of the ZEN degradation efficiency. Overall, the supernatant degraded over 75% of ZEN within a concentration range of 1 μg / mL to 40 μg / mL.

[0117] It is noteworthy that the supernatant still retained the ability to degrade ZEN after boiling, with a degradation rate of 88.45% ± 0.68%, indicating the presence of heat-resistant active components. Proteinase K and SDS treatment significantly reduced the supernatant's ZEN-degrading activity, with SDS having the greatest effect on the supernatant's detoxification activity, followed by proteinase K. In particular, SDS completely inhibited ZEN degradation. The results suggest that the degradation in the supernatant may be caused by one or more heat-resistant proteases ( Figure 2 E).

[0118] like Figure 2As shown in Figure F, the degradation rate significantly increased with increasing ZEN incubation time. The degradation effect of the supernatant on ZEN increased with time. Meanwhile, after 24 hours, the degradation rate of ZEN in the supernatant stabilized, reaching over 86%. However, supernatant analysis showed that the main degradation activity originated from enzyme-like proteins, which typically exhibit rapid catalysis. Studies have also explored short-term, high-efficiency enzymes.

[0119] 2.2 Genome-wide identification of proteins involved in ZEN degradation

[0120] The whole genome sequencing of Bacillus velezinis B.26 showed that it has a 3.97Mb circular chromosome (GC content: 46.5%). After sequencing the whole genome of strain B.26 and performing functional annotation using various tools, 4030 protein-coding genes were identified ( Figure 3 A), it is noteworthy that 12.3% of the annotated genes were enriched in oxidoreductase activity. Meanwhile, strain B.26 was identified as Bacillus velezensis ( Figure 3 B).

[0121] 2.3 Selection of CotA and Prx for ZEN degradation

[0122] Silva et al. reported that laccases from different sources can degrade a variety of mycotoxins. Previous studies have confirmed the mycotoxin degradation ability of Bacillus licheniformis CotA laccase (GenBank: QAX90317.1). To identify novel, unreported laccases for mycotoxin detoxification, a bioinformatics analysis based on NCBI BLAST was performed. The results showed that the sequence identity between CotA laccase and its homologs was 66.12% ( Figure 4 A). Parallel analysis of Prx revealed that it has a 42.65% homology with the peroxide reductase of Acinetobacter nosocomialis Y1, and both enzymes have potential mycotoxin degradation capabilities ( Figure 4 B).

[0123] The putative functional domains of CotA and Prx were systematically characterized by an integrated bioinformatics workflow, using InterProScan for primary sequence analysis. For CotA, three conserved multi-copper oxidase domains were identified. Figure 4 The catalytic structure of laccase in C is consistent. Figure 4 D, SMART domain annotation revealed that Prx contains a typical thioredoxin-dependent peroxide reductase domain (PF00578; 96% coverage), consistent with its putative role in mycotoxin degradation via a redox-mediated mechanism.

[0124] 2.4 Characteristics of CotA and Prx

[0125] The Prx and CotA genes were inserted into the pET-28a plasmid to construct the recombinant plasmids pET-28a-prx and pET-28a-cotA. These plasmids were transformed into Escherichia coli BL21 cells, and the modified strains were then treated to produce Prx and CotA proteins within a 16-hour induction period. The theoretical molecular weights of Prx and CotA were calculated to be 22 kDa and 62 kDa, respectively, and SDS-PAGE analysis showed electrophoretic mobilities consistent with these predictions ( Figure 5 A and 5B). Heterologous expression in E. coli produced 420 mg / L of Prx and 710 mg / L of CotA.

[0126] The detailed docking scores and hydrogen bonding parameters are listed in Table 2. The higher binding affinity (-8.2 kcal / mol) and extensive hydrogen bonding network indicate that the CotA complex has a higher stability compared with Prx. Figure 5 As shown in C, molecular docking showed that ZEN formed six hydrogen bonds with the catalytic residues of CotA. Figure 5 The interaction between ZEN and Prx in D involves three hydrogen bonds, and these hydrogen-bonding residues critically stabilize the substrate orientation within the enzyme active site.

[0127] Table 2 Molecular docking scores and hydrogen bond distances between enzymes and zearalenone (ZEN)

[0128]

[0129] 2.4 Degradation characteristics of ZEN by CotA and Prx

[0130] like Figure 6 As shown in A, the degradation rate of CotA reached 90% within 6 hours. When the incubation time was extended, the degradation rate remained unchanged at 8 hours. Therefore, 6 hours is the time when CotA reaches its peak degradation rate. After 6 hours of observation, it was found that the degradation rate of Prx was also the highest at 6 hours, which was 46.51% ( Figure 6 F).

[0131] The detoxification ability of purified CotA and Prx to ZEN was quantitatively evaluated under a concentration gradient (1-40 μg / mL). Figure 6 As shown in B, CotA exhibited excellent detoxification efficacy, with a degradation rate of over 80% for ZEN at concentrations below 10 μg / mL within 6 hours. In contrast, Prx had limited catalytic efficiency, achieving only 50% degradation at the lowest concentration (1 μg / mL). Figure 6 G) For Prx, extended time points were attempted, but complete detoxification of the mycotoxins was still not possible.

[0132] Enzymatic characterization showed that the optimum temperature for both was 70℃( Figure 6 C and 6H), both enzymes retained more than 60% residual activity at temperatures of 70°C and above. After incubation at pH 8.0 for 6 hours, CotA retained 100% enzyme activity, while Prx showed optimal function at pH 11.0 ( Figure 6 D and 6I), both catalysts showed remarkable alkaline stability. Comparative analysis confirmed that the thermal adaptation profile of CotA was consistent with that of typical bacterial laccases. Compared with their neutral pH-adapted counterparts, the different pH optima of the two biocatalysts indicate enhanced functional robustness under alkaline conditions, indicating that they are more suitable for industrial biocatalytic processes requiring extreme pH tolerance.

[0133] The enzyme activities of CotA and Prx at different metal ion concentrations were systematically evaluated using standardized biochemical assays, such as Figure 6 As shown in E and 6J, Mn 2+ It showed significant inhibitory effects on both enzymes, resulting in an almost complete loss of their catalytic efficiency. 2 + 、Na + , K + and Li+ ions enhanced the degradation activity of CotA. 2+ and Mg 2+ showed high sensitivity, resulting in 96.77% and 94.81% activity loss at 10 mM concentration, respectively. 2+ and Zn 2+ The results showed remarkable sensitivity, with only 0.69% and 3.53% residual activity remaining under the same experimental conditions. The control assay without metal supplementation maintained full enzyme function throughout the analysis.

[0134] 2.6 LC / MS Analysis of ZEN Degradation Products

[0135] LC / MS analysis showed that ZEN eluted at 13.319 minutes with a mass-to-charge ratio (m / z) of 317.13907 [MH]-, corresponding to a molecular weight (MW) of 318.1464, and a molecular formula of C 18 H 22 O5. The mass spectrum of the blank control group without adding degradation enzyme is as follows Figure 7 As shown in A and 8(B), the secondary mass spectrum of ZEN is as follows Figure 10 (A) To eliminate the effect of the degradation enzyme, the mass spectrum of the system with only the degradation enzyme added is shown in Figure 7B and 9(A). Compared with the control group, the amount of ZEN residue was significantly reduced after enzyme treatment with CotA and Prx, as evidenced by the reduction of peak area. It is worth noting that the degradation efficiency of CotA is better than that of Prx.

[0136] Crucially, no levels of known toxic metabolites (e.g., α-ZEL, β-ZEL, α-ZAL, or β-ZAL) were detected after treatment ( Figure 8 (A) and 10 (B)), indicating that the enzymatic degradation of both catalysts proceeds through alternative pathways that avoid the formation of these specific harmful compounds. In this group, a distinct chromatographic peak (m / z: 291.1601 [MH]-; MW: 292.1674; peak area: 1.97 × 10 6 ), and the metabolite was identified as C by structural annotation using Compound Discoverer software. 17 H 24 O4, which is consistent with the previous report that the lactone ring cleavage is the detoxification mechanism of ZEN. The estrogenic activity of ZEN mainly comes from its oxygen-containing macrocyclic lactone structure. The detoxification product (C 17 H 24 O4) has been shown to have an estrogenic potency ≥1,000-fold lower than that of ZEN and has no adverse morphological effects on the reproductive system of prepubertal gilts, emphasizing the safety of CotA-mediated degradation.

[0137] like Figure 9 (B) and Figure 11 As shown, in the Prx-treated group, a hydrophilic metabolite (elution time: 12.08 min; m / z: 223.0975 [MH] - MW: 224.1048; Peak area: 2.43×10 6 ), structural analysis specifies its molecular formula as C 12 H 16 O4, suggesting modifications involving ketone reduction, hydroxylation, or ring-opening pathways. Although the precise structure of this compound (m / z 223.0975) has not been characterized in the literature, its similarity to ZEN-derived metabolites reported in the Bacillus subtilis system suggests a novel detoxification pathway. Notably, the absence of an intact lactone ring is consistent with established mechanisms for mitigating ZEN's estrogenic activity.

[0138] The major degradation product C from CotA was predicted 17 H 24 O4 and C from Prx 12 H 16 The structure of O4 is as follows Figure 12Overall, these findings suggest that CotA and Prx mediate ZEN detoxification through distinct but safe pathways without generating harmful intermediates.

[0139] 3. Analysis of test results

[0140] As a ubiquitous mycotoxin produced by Fusarium spp., ZEN (Zen) poses a significant global food safety risk due to its estrogenic effects and thermostability in agricultural products. The coexistence of ZEN with other mycotoxins exacerbates health risks through synergistic toxicity, particularly in cereal-based products. Traditional physical and chemical detoxification methods often lack specificity or risk the generation of secondary contaminants. Enzymatic degradation, on the other hand, offers a targeted pathway with substrate specificity, safety, efficiency, and environmental compatibility, generating non-toxic or low-toxic metabolites. Although microbial degradation has achieved 70–90% reductions in ZEN in reported cases, its practical application faces three obstacles: pH incompatibility, thermostability, and metabolite risks. Fungal laccases have an optimal pH range of 3.0–5.0 and lose activity in alkaline feed matrices. During feed processing, such as corn gelatinization (70–90°C) and corn puffing, temperatures of 60–90°C are encountered, and many enzymes are denatured in this environment. Bacterial reductases frequently convert ZEN to α-Zal. In this study, we identified an extracellular ZEN-degrading enzyme secreted by Bacillus velezinis B.26 from a laboratory strain collection. This strain B.26 could almost completely degrade approximately 91.64% ± 0.24% of 2 μg / mL ZEN within 24 h under the optimal conditions of 70°C and pH 10.0.

[0141] Boiling treatment had no significant effect on the ZEN-degrading ability of the supernatant of Bacillus velezensis B.26, confirming the thermostability of its enzyme components, which is consistent with reports of thermostable ZEN-degrading enzymes from Acinetobacter SM04. Notably, SDS significantly inhibited the activity of SM04, a pattern consistent with the findings of this study, leading to the hypothesis that a surfactant-sensitive protease is the key degrader in SM04.

[0142] Heterologous expression of CotA and Prx in Escherichia coli BL21 yielded enzymes with exceptional thermoelasticity. Both CotA and Prx retained over 60% of their relative activity at elevated temperatures, outperforming their mesophilic counterparts, such as the peroxide reductase derived from Acinetobacter spp. Their optimal activity at 70°C makes them practical candidates for feed pelleting (55-80°C) and grain sterilization.

[0143] Comparative analysis revealed that under acidic conditions (pH 3.0-5.0), the fungal laccase PpLac1 was able to degrade 70.84% ​​of 2 μg / mL ZEN within 24 hours, while in this study, CotA achieved complete degradation within 6 hours at 70°C and pH 10.0, demonstrating that CotA outperformed the fungal laccase in degradation of ZEN. The bacterial CotA laccase required immobilization to achieve 90% degradation efficiency at 80°C, compared to 70% for the free enzyme, indicating that enzyme stabilization is crucial for maintaining catalytic performance under the extreme thermal conditions typical of feed and pet food sterilization. This alkali and heat resistance contrasts sharply with the 70% efficiency of Bacillus licheniformis CotA at 60°C and neutral pH. After incubation at 70°C for 1 hour, CotA retained 80% of its enzyme activity, showing better thermal stability than Aspergillus oryzae Laccase 2 (AoLac2), which lost 40% of its activity at 60°C. These results indicate that CotA meets the industrial requirements for resistance to thermal denaturation.

[0144] The optimum pH for CotA is 8.0, while that for Prx is 11.0, reflecting different catalytic mechanisms. The alkaline preference of CotA is associated with structural stability under extreme conditions, reflecting the characteristics of bacterial laccases, while Prx exhibits optimal activity at neutral pH, consistent with the spectrum of porin-related enzymes. The metal ion effect reflects the homologous system, Mn 2+ strongly inhibited both enzymes, while Cu 2+ Enhanced CotA activity may promote electron transfer during oxidation. 2+ The degradation efficiency of CotA was enhanced, and combined with the observed Cu 2+ The effect of the fermentation supernatant on the detoxification activity of the fermentation supernatant showed that CotA was the main enzyme responsible for ZEN degradation in the supernatant. 2+ It can significantly enhance the degradation ability of enzymes and bacteria, such as the degradation of ZEN by porins and peroxidase, and the degradation of AFB1 by BADE, which indicates that Cu 2+ It may act as an electron transporter within the enzyme molecule and participate in the enzyme-catalyzed reaction. This conserved regulatory mechanism in thermostable oxidoreductases warrants molecular dynamics simulations to elucidate the structural determinants of CotA thermal stability.

[0145] Based on LC-MS / MS analysis, it was hypothesized that the degradation product of ZEN in this study under CotA treatment might be C 17 H 24 O4, C under Prx treatment 12 H 16O4. The retention time of the degradation peak is earlier than that of ZEN, indicating that these metabolites are hydrophilic. ZEN degradation mainly involves hydroxyl ketone reduction, double bond cleavage, hydroxylation, methylation, sulfonation, glycosylation and lactone ring opening. The CotA-derived products are structurally consistent with the findings of the prior art, while the Prx-derived products are consistent with existing reports. However, this study proposes that these only represent the main degradation products, and both enzymes achieve detoxification by destroying the oxygenated lactone ring (the key pharmacophore of estrogenicity).

[0146] 2. Extended test:

[0147] In another embodiment, a method for fermenting and degrading zearalenone by Bacillus velezinii for alkaline feed processing comprises the following steps:

[0148] Step 1: crushing the zearalenone-contaminated feed raw material to a particle size of 0.5-1.0 mm, and mixing it with sterile water at a mass ratio of 1:5 to obtain a pretreated feed suspension;

[0149] Step 2: Inoculate Bacillus velezensis CGMCC No. 34475 into an activation medium containing 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride, adjust the initial pH to 8.5 with sodium bicarbonate buffer, and incubate the culture in a 37°C constant temperature shaker at 180 rpm for 12 hours;

[0150] Step 3: Inoculate the activated bacterial solution into the pretreated feed suspension at a volume ratio of 10%, add 0.5% sucrose as an induced carbon source, and use 5% sodium carbonate solution to adjust the initial pH of the fermentation system to 10.5;

[0151] Step 4: The fermentation system was placed in a sealed fermentation tank, the temperature in the fermentation tank was controlled at 37°C, sterile air was introduced to maintain the dissolved oxygen concentration at 30%-40%, and the pH of the fermentation liquid was monitored in real time by an online pH probe and automatically supplemented with a 5% mass concentration of sodium carbonate solution to maintain the pH of the fermentation liquid in the range of 10.5-11;

[0152] After 24 hours of fermentation under continuous stirring at 180 rpm, the fermentation was terminated;

[0153] Step 5: After solid-liquid separation of the fermented material, the solid material is dried at 60° C. with hot air to a moisture content of less than 12%, thereby obtaining a finished feed product after degradation treatment.

[0154] In the above embodiment, in the crushing process, the crusher can be a hammer crusher or a blade crusher, and the screen aperture can be selected from three specifications: 0.6mm, 0.8mm or 1.0mm. The mixing equipment can be a vertical stirring tank or a horizontal mixer, and the linear speed of the stirring blade is controlled at 2-3m / s. The pretreatment workshop can be equipped with a vibrating screen for particle size classification, and the crushed material enters the mixing section through a pneumatic conveying system. Sterile water preparation can use a reverse osmosis pure water unit combined with an ultraviolet sterilization device, and the conductivity is controlled at below 5μS / cm. The pretreated suspension is transported to a temporary storage tank by a screw pump. The tank body material can be selected from 316L stainless steel and equipped with a jacket insulation system.

[0155] A magnetic stirring dissolution tank can be used for the preparation of activated culture medium, and the dissolution temperature is controlled at 45-50°C. An online pH adjustment system is used for the preparation of sodium bicarbonate buffer, and the buffer concentration can be selected from 50mM, 100mM or 150mM. A double-layer constant temperature shaker can be used for shaking culture, and the tray isolation pad is made of silicone material. The seed tank inoculation system can be equipped with a peristaltic pump, and the pump tube material is platinum-sulfurized silicone. The sodium carbonate solution storage tank can be equipped with a heating and insulation system, and the solution temperature is maintained at 35-40°C. The fermentation tank feeding system is equipped with an online weighing module, and the weighing accuracy reaches ±0.5%.

[0156] Dissolved oxygen control can be achieved with a membrane aerator with a pore size distribution of 0.2-0.5 μm, and the aeration rate is adjusted via a mass flow meter. The pH control system is equipped with a dual-channel feed pump, and the feed line is equipped with a one-way check valve. A vibrating fluidized bed dryer can be used for the drying process, and the air inlet filter should use H13 high-efficiency filter material. The online monitoring system integrates a turbidity sensor and a near-infrared spectrometer, with a data collection interval set to 5 minutes. The solid-liquid separation section can be equipped with a horizontal spiral centrifuge with a differential speed adjustment range of 2-15 rpm and a filter mesh size of 200-400 mesh.

[0157] This implementation utilizes specific particle size reduction to improve bacterial-enzyme contact efficiency, staged pH control to maintain bacterial activity, a composite aeration system to optimize oxygen transfer, and multi-parameter online monitoring to ensure process stability. The final product meets industrial production requirements in terms of toxin degradation rate, nutrient retention, and processing cost control.

[0158] In another embodiment, the activation culture of Bacillus velezinis adopts a staged pH adaptation acclimation operation, and the specific steps include:

[0159] Under the condition that the initial pH of the activation medium was adjusted to 8.5, the culture was carried out in a constant temperature shaker at 37°C and 180 rpm for 8 hours;

[0160] The pH of the culture system was gradually raised to 10.0 by aseptically adding 5% sodium carbonate solution, and the culture temperature was maintained at 37°C for another 4 hours.

[0161] During the pH increase process, when the OD600 value of the culture solution dropped by more than 15%, 0.1% trehalose was immediately added as a cell membrane protectant.

[0162] After completing the two-stage culture, the bacterial solution was inoculated back into fresh activated culture medium at a volume ratio of 15%, and cultured for another 3 hours at pH 10.0 to obtain an alkali-resistant acclimated strain.

[0163] In the above embodiment, the pH value in the initial culture stage can be set to three gradients of 8.3, 8.5 or 8.7, and the culture time can be selected to be 7 hours, 8 hours or 9 hours. The constant temperature shaker can use a double-layer or triple-layer tray structure, and the tray material can be polycarbonate or 304 stainless steel. The pH adjustment device can be equipped with a peristaltic pump, the pump head flow adjustment range is 0.1-5mL / min, and the volume of the sodium carbonate solution storage tank can be 1.2-1.5 times the volume of the culture solution. The culture container can be installed with a side observation window and equipped with an LED backlighting system. In the pH increase stage, it is gradually adjusted to three gradients of 9.5, 9.8, and 10.0 at intervals of 1 hour three times, and the adjustment amount each time does not exceed 0.5pH units.

[0164] The trehalose dosing system can be integrated into the top of the culture vessel, and the dosing line can be equipped with a microinjection pump with a flow rate accuracy of ±0.5 μL / min. The OD600 monitor can be equipped with an online fiber optic probe, with a detection wavelength bandwidth set to ±2 nm. The protective agent storage tank can be equipped with a magnetic stirrer to maintain solution homogeneity, with the stirring speed controlled at 100-150 rpm. The dosing procedure is triggered when the OD600 value drops by 16%, 18%, or 20%. Dosing can be completed in two 30-minute intervals, with each dose accounting for 50% of the total required amount.

[0165] An automated pipetting workstation can be used for reconnection operations, and the pipetting head can be configured with an 8-channel or 12-channel structure. Fresh culture medium can be sterilized in a high-pressure steam sterilizer at 121°C for 20 minutes. During the intensive culture phase, the stirring propeller can be replaced with a pitched-blade propeller, with the ratio of the propeller diameter to the tank diameter controlled at 0.3-0.4. The culture vessel exhaust system can be equipped with a condensation reflux device, with the condensation temperature set at 4-6°C. For bacterial strain preservation, the glycerol tube freezing method can be used, with a storage temperature of -80°C and a final glycerol concentration of 15%.

[0166] This implementation reduces bacterial stress responses through gradient pH regulation, supplements with dynamic protectants to maintain cell membrane stability, and enhances inoculation to screen for highly resistant strains. This process maintains bacterial activity and metabolic efficiency, improves bacterial adaptability in extremely alkaline environments, and provides high-quality inoculum for subsequent large-scale fermentation.

[0167] In another embodiment, in step 3, the fermentation induction operation adopts a phased supply strategy of a composite carbon source, and the specific steps include:

[0168] During the fermentation period of 0-12 hours, the stirring speed was maintained at 180 rpm, and sucrose with a mass concentration of 0.3% was continuously added as the basic carbon source;

[0169] During the fermentation period of 12-24 hours, a complex of 0.1% inulin and 0.05% sodium citrate was added simultaneously, wherein the mass ratio of inulin to sodium citrate was 2:1;

[0170] When the viscosity of the fermentation broth rises to 2000 cP, a 0.2% mass concentration of β-cyclodextrin solution is pulse-injected into the fermentation broth, and the injection volume each time is 1% of the total volume of the fermentation broth;

[0171] The cumulative amount of β-cyclodextrin added does not exceed 0.1% of the dry matter mass of the fermentation liquid;

[0172] During the fermentation process, the ATP content of the bacteria was detected every 6 hours. When the ATP content was lower than 5 μmol / gDCW, the carbon source supply was immediately terminated and the cooling program was started.

[0173] In the above embodiment, during the 0-12 hour period, the basal carbon source concentration can be set to three gradients of 0.25%, 0.3%, or 0.35%. The continuous dosing equipment can use a peristaltic pump in conjunction with an online static mixer, and the pumping flow error is controlled within ±2%. The sucrose raw material can be food-grade white sugar with a purity of ≥99.5%. During the 12-24 hour period, the ratio of inulin to sodium citrate in the composite carbon source can be adjusted to 1.5:1, 2:1, or 2.5:1. The inulin raw material can be chicory root extract with a degree of polymerization DP of ≥10. Sodium citrate can be pharmaceutical-grade dihydrate with a purity of ≥99.0%. The carbon source addition port can be located 15 cm above the aeration plate at the bottom of the fermenter.

[0174] Viscosity can be measured using an online rotational viscometer with a measurement range of 500-3000 cP. The probe should be installed at a radius of 1 / 3 of the tank wall. The β-cyclodextrin solution storage tank can be equipped with a constant-temperature water bath to maintain a solution temperature of 30°C. A solenoid diaphragm valve with a response time of ≤0.5 seconds can be used as the pulse injection valve. Hydroxypropyl-β-cyclodextrin with a degree of substitution of 0.6-0.9 can be used as the cyclodextrin raw material. A Y-type filter with a pore size of 10 μm can be installed in the injection line. The injection volume can be controlled to 0.8%, 1.0%, or 1.2% of the total fermentation broth volume, with an injection interval of ≥15 minutes.

[0175] ATP detection can be performed using a bioluminescence detector. The sampling port can be located on the sampling valve in the middle of the fermenter, and the sampling frequency can be set to every 5, 6, or 7 hours. Microbial dry weight can be determined using a microwave dryer in combination with an electronic balance, with a weighing accuracy of 0.1 mg. The cooling process can be performed using a plate heat exchanger with an ethylene glycol-water solution as the cooling medium, with a cooling rate controlled at 0.5-1°C / min. The process control cabinet can be integrated with a PID controller, with a temperature control accuracy of ±0.2°C. The data acquisition system can be configured with a 4-20mA signal conversion module with a sampling period of 100ms.

[0176] This implementation scheme utilizes phased carbon source supply to match bacterial metabolic needs, viscosity control to maintain dissolved oxygen transfer efficiency, and dynamic ATP monitoring to promptly terminate abnormal fermentation. A complex carbon source combination promotes the synthesis of secondary metabolites, while the addition of cyclodextrin enhances substrate contact efficiency. Interlocked control of all process parameters ensures process stability, effectively enhancing the strain's toxin degradation efficiency in alkaline environments.

[0177] <Example 1>

[0178] Experimental Materials

[0179] Zearalenone-contaminated feed (initial concentration 1.5 mg / kg), Bacillus velezensis CGMCC No.34475, trypsin, yeast extract, sodium chloride, sodium bicarbonate, sodium carbonate, and sucrose.

[0180] Experimental process

[0181] 1. Preprocessing

[0182] The contaminated feed was crushed to a particle size of 0.8 mm using a hammer mill, mixed with sterile water (conductivity ≤ 5 μS / cm) at a mass ratio of 1:5, and stirred for 30 min to obtain a suspension (pH 7.0).

[0183] 2. Bacteria activation

[0184] Activation medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, adjusted to pH 8.5 with 100 mM sodium bicarbonate buffer.

[0185] After inoculation, the culture was shaken at 37°C and 180 rpm for 12 hours without pH adaptation.

[0186] 3. Fermentation Culture

[0187] The bacterial liquid was inoculated into the suspension at a 10% inoculum volume, and 0.5% sucrose and 5% sodium carbonate were added to adjust the initial pH to 10.5.

[0188] Fermentation was carried out in a closed fermentation tank at 37°C for 24 hours, with dissolved oxygen maintained at 30%-40%, and sodium carbonate was automatically added to control the pH to 10.5-11.0, with a stirring speed of 180 rpm.

[0189] 4. Post-processing

[0190] The fermentation broth was separated by a horizontal spiral centrifuge (3000 rpm, 15 minutes), and the solid was dried with hot air at 60°C to a moisture content of ≤12%.

[0191] Detection method

[0192] 1. Toxin detection

[0193] 5 g of the dried sample was extracted with acetonitrile-water (84:16) by ultrasonication for 30 minutes, and then filtered through a 0.22 μm filter membrane after centrifugation.

[0194] HPLC conditions: C18 column (4.6×250 mm), mobile phase: acetonitrile-water-methanol (46:46:8), flow rate: 1.0 mL / min, fluorescence detection (Ex 274 nm, Em 440 nm).

[0195] 2. Bacterial activity detection

[0196] The OD600 value was determined by a spectrophotometer, and the ATP content was detected using a bioluminescence detection kit.

[0197] Experimental results

[0198] The residual concentration of zearalenone in zearalenone is 0.45 mg / kg, and the degradation rate is 70%.

[0199] The OD600 peak value was 4.2 after 12 hours of fermentation, and then dropped to 2.5 in the later stage. The pH fluctuated between 10.3 and 11.2.

[0200] <Example 2>

[0201] Experimental Adjustment

[0202] 1. Increase pH acclimation during the bacterial activation stage

[0203] The pH was 8.5 for the first 8 hours, and 5% sodium carbonate was gradually added to raise the pH to 10.0 for the next 4 hours. When the OD600 dropped by more than 15%, 0.1% trehalose was added.

[0204] The bacterial solution was inoculated back into fresh culture medium at pH 10.0 and incubated for 3 hours.

[0205] 2. Fermentation parameter optimization

[0206] The initial pH was adjusted to 10.5, and 0.1% trehalose was added to protect the bacteria.

[0207] Experimental results

[0208] The residual concentration of toxin was 0.28 mg / kg, and the degradation rate was 81.3%.

[0209] The OD600 peak was 6.5, and the pH was stable at 10.5-10.8 throughout the process, with no sudden drop in bacterial activity.

[0210] <Example 3>

[0211] Experimental Adjustment

[0212] 1. Carbon source supply in stages

[0213] 0.3% sucrose was continuously added from 0 to 12 hours, and 0.1% inulin (DP≥10) and 0.05% sodium citrate were added simultaneously from 12 to 24 hours.

[0214] When the viscosity of the fermentation broth reached 2000 cP, a pulse injection of 0.2% β-cyclodextrin solution (total amount ≤ 0.1% dry matter) was performed.

[0215] 2. Dynamic metabolic monitoring

[0216] The ATP content was detected every 6 h, and the fermentation was terminated when it was lower than 5 μmol / gDCW.

[0217] Experimental results

[0218] The residual concentration of toxin is 0.12 mg / kg, and the degradation rate is 92%.

[0219] After 18 hours of fermentation, the ATP content was stabilized at 8.2 μmol / gDCW, the dissolved oxygen was maintained at 25%-35%, and the pH was 10.5-10.7 throughout the process.

[0220] Comparative Example 1

[0221] Experimental conditions

[0222] Use untamed bacteria, single-stage fermentation at pH 10.5, 0.3% glucose carbon source throughout the process, and no complex carbon source or β-cyclodextrin.

[0223] Experimental results

[0224] The residual concentration of toxin is 1.02 mg / kg, and the degradation rate is 32%.

[0225] After 6 hours of fermentation, the OD600 dropped to 1.8, the pH rose out of control to 11.5, and the bacteria were inactivated.

[0226] Experiments revealed that the degradation rate of Bacillus velezensis CGMCC No. 34475 was 70% without acclimation, with a significant decrease in bacterial activity in the later stages. Through pH acclimation and trehalose protection, the degradation rate increased to 81.3%, enhancing stability. Combining a composite carbon source with dynamic regulation, the degradation rate reached 92%, with stable metabolic activity throughout. Traditional methods, due to the strain's alkali intolerance and a single carbon source, only resulted in a degradation rate of 32%. Phased pH acclimation, the supply of a composite carbon source, and dynamic monitoring significantly improved the strain's toxin degradation efficiency in alkaline environments.

[0227] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A thermophilic and alkaline resistant Bacillus velez strain, characterized in that: Its classification name is Bacillus velezensis. The Bacillus velezensis strain is preserved as Bacillus velezensis B.26 and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No.34475.

2. The use of the Bacillus velez according to claim 1 in degrading zearalenone, wherein the use is carried out at a temperature of 50-80°C and a pH of 7.0-12.0, and the reaction time is 1-24 hours, so that zearalenone is degraded into a low-toxic metabolite C 17 H 24 O4 and C 12 H 16 O4, and no α-zearalenol or β-zearalenol was produced.

3. Use of the Bacillus Velez subtilis according to claim 1 in the preparation of an enzyme for degrading zearalenone.

4. The use according to claim 3, characterized in that The enzyme for degrading zearalenone is a recombinant CotA laccase, the amino acid sequence of which is shown in SEQ ID NO: 3, and the nucleotide sequence of the recombinant CotA laccase is shown in SEQ ID NO:

1.

5. The use according to claim 3, characterized in that The enzyme for degrading zearalenone is a recombinant peroxide reductase Prx, the amino acid sequence of which is shown in SEQ ID NO: 4, and the nucleotide sequence of the recombinant peroxide reductase Prx is shown in SEQ ID NO:

2.

6. A method for degrading zearalenone, characterized in that: The following steps are involved: contacting the recombinant CotA laccase according to claim 4 or the recombinant peroxide reductase Prx according to claim 5 with a substrate containing zearalenone; The reaction is carried out at a temperature of 50-80° C. and a pH of 7.0-12.0 for 1-24 hours to degrade ZEN into low-toxic metabolites.

7. A detoxification agent composition, characterized in that It comprises the recombinant CotA laccase according to claim 4 and / or the recombinant Prx according to claim 5, and a pharmaceutically or feed-acceptable carrier; the detoxification agent composition is used for the degradation of zearalenone in food or feed, wherein the carrier is calcium alginate microspheres, montmorillonite-based materials or nanoparticles, which are used for enzyme immobilization or sustained release.

8. A method for fermenting and degrading zearalenone by Bacillus velezensis for alkaline feed processing, characterized in that: The following steps are involved: Step 1: crushing the zearalenone-contaminated feed raw material to a particle size of 0.5-1.0 mm, and mixing it with sterile water at a mass ratio of 1:5 to obtain a pretreated feed suspension; Step 2: Inoculate Bacillus velezensis CGMCC No. 34475 into an activation medium containing 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride, adjust the initial pH to 8.5 with sodium bicarbonate buffer, and incubate the culture in a 37°C constant temperature shaker at 180 rpm for 12 hours; Step 3: Inoculate the activated bacterial solution into the pretreated feed suspension at a volume ratio of 10%, add sucrose with a mass concentration of 0.3%-0.5% as an induced carbon source, and use a sodium carbonate solution with a mass concentration of 5% to adjust the initial pH of the fermentation system to 10.5; Step 4: The fermentation system was placed in a sealed fermentation tank, the temperature in the fermentation tank was controlled at 37°C, sterile air was introduced to maintain the dissolved oxygen concentration at 30%-40%, and the pH of the fermentation liquid was monitored in real time by an online pH probe and automatically supplemented with a 5% mass concentration of sodium carbonate solution to maintain the pH of the fermentation liquid in the range of 10.5-11; After 24 hours of fermentation under continuous stirring at 180 rpm, the fermentation was terminated; Step 5: After solid-liquid separation of the fermented material, the solid material is dried at 60° C. with hot air to a moisture content of less than 12%, thereby obtaining a finished feed product after degradation treatment.

9. The method for fermenting and degrading zearalenone by Bacillus velezensis for alkaline feed processing according to claim 8, characterized in that: The activation culture of Bacillus Velez is carried out by a staged pH adaptation acclimation operation, the specific steps include: Under the condition that the initial pH of the activation medium was adjusted to 8.5, the culture was carried out in a constant temperature shaker at 37°C and 180 rpm for 8 hours; The pH of the culture system was gradually raised to 10.0 by aseptically adding 5% sodium carbonate solution, and the culture temperature was maintained at 37°C for another 4 hours. During the pH increase process, when the OD600 value of the culture solution dropped by more than 15%, 0.1% trehalose was immediately added as a cell membrane protectant. After completing the two-stage culture, the bacterial solution was inoculated back into fresh activated culture medium at a volume ratio of 15%, and cultured for another 3 hours at pH 10.0 to obtain an alkali-resistant acclimated strain.

10. The method for fermenting and degrading zearalenone by Bacillus velezensis for alkaline feed processing according to claim 9, characterized in that: In step 3, the fermentation induction operation adopts a phased supply strategy of a composite carbon source, and the specific steps include: During the fermentation period of 0-12 hours, the stirring speed was maintained at 180 rpm, and sucrose with a mass concentration of 0.3% was continuously added as the basic carbon source; During the fermentation period of 12-24 hours, a complex of 0.1% inulin and 0.05% sodium citrate was added simultaneously, wherein the mass ratio of inulin to sodium citrate was 2:1; When the viscosity of the fermentation broth rises to 2000 cP, a 0.2% mass concentration of β-cyclodextrin solution is pulse-injected into the fermentation broth, and the injection volume each time is 1% of the total volume of the fermentation broth; The cumulative amount of β-cyclodextrin added does not exceed 0.1% of the dry matter mass of the fermentation liquid; During the fermentation process, the ATP content of the bacteria was detected every 6 hours. When the ATP content was lower than 5 μmol / gDCW, the carbon source supply was immediately terminated and the cooling program was started.

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